Methods for in vivo delivery of substantially water insoluble pharmacologically active agents and compositions useful therefor

ABSTRACT

In accordance with the present invention, there are provided compositions for the in vivo delivery of substantially water insoluble pharmacologically active agents (such as the anticancer drug taxol) in which the pharmacologically active agent is delivered in a soluble form or in the form of suspended particles. In particular, the soluble form may comprise a solution of pharmacologically active agent in a biocompatible dispersing agent contained within a protein walled shell. Alternatively, the protein walled shell may contain particles of taxol. In another aspect, the suspended form comprises particles of pharmacologically active agent in a biocompatible aqueous liquid.

This application is a divisional of application Ser. No. 08/023,698,filed Feb. 22, 1993. now U.S. Pat. No. 5,439,686.

The present invention relates to in vivo delivery of substantially waterinsoluble pharmacologically active agents (e.g., the anticancer drugtaxol). In one aspect, the agent is dispersed as a suspension suitablefor administration to a subject, or is dissolved in a suitablebiocompatible liquid. In another aspect, water insolublepharmacologically active agents (e.g., taxol) are encased in a polymericshell formulated from a biocompatible polymer. The polymeric shellcontains particles of pharmacologically active agent, and optionally abiocompatible dispersing agent in which pharmacologically active agentcan be either dissolved or suspended.

BACKGROUND OF THE INVENTION

Taxol is a natural product first isolated from the Pacific Yew tree,Taxus brevifolia, by Wani et al. [J. Am. Chem. Soc. Vol. 93:2325(1971)]. Among the antimitotic agents, taxol, which contains a diterpenecarbon skeleton, exhibits a unique mode of action on microtubuleproteins responsible for the formation of the mitotic spindle. Incontrast with other antimitotic agents such as vinblastine orcolchicine, which prevent the assembly of tubulin, taxol is the onlyplant product known to inhibit the depolymerization process of tubulin,thus preventing the cell replication process.

Taxol, a naturally occurring diterpenoid, has been shown to havesignificant antineoplastic and anticancer effects in drug-refractoryovarian cancer. Taxol has shown excellent antitumor activity in a widevariety of tumor models such as the B16 melanoma, L1210 leukemias, MX-1mammary tumors, and CS-1 colon tumor xenografts. Several recent pressreleases have termed taxol as the new anticancer wonder-drug. Indeed,taxol has recently been approved by the Federal Drug Administration fortreatment of ovarian cancer. The poor aqueous solubility of taxol,however, presents a problem for human administration. Indeed, thedelivery of drugs that are inherently insoluble or poorly soluble in anaqueous medium can be seriously impaired if oral delivery is noteffective. Accordingly, currently used taxol formulations require acremaphore to solubilize the drug. The human clinical dose range is200-500 mg. This dose is dissolved in a 1:1 solution ofethanol:cremaphore and diluted to one liter of fluid givenintravenously. The cremaphore currently used is polyethoxylated castoroil.

In phase I clinical trials, taxol itself did not show excessive toxiceffects, but severe allergic reactions were caused by the emulsifiersemployed to solubilize the drug. The current regimen of administrationinvolves treatment of the patient with antihistamines and steroids priorto injection of the drug to reduce the allergic side effects of thecremaphore.

In an effort to improve the water solubility of taxol, severalinvestigators have modified its chemical structure with functionalgroups that impart enhanced water-solubility. Among them are thesulfonated derivatives [Kingston et al., U.S. Pat. No. 5,059,699(1991)], and amino acid esters [Mathew et al., J. Med. Chem. Vol.35:145-151 (1992)] which show significant biological activity.Modifications to produce a water-soluble derivative facilitate theintravenous delivery of taxol dissolved in an innocuous carrier such asnormal saline. Such modifications, however, add to the cost of drugpreparation, may induce undesired side-reactions and/or allergicreactions, and/or may decrease the efficiency of the drug.

Microparticles and foreign bodies present in the blood are generallycleared from the circulation by the `blood filtering organs`, namely thespleen, lungs and liver. The particulate matter contained in normalwhole blood comprises red blood cells (typically 8 microns in diameter),white blood cells (typically 6-8 microns in diameter), and platelets(typically 1-3 microns in diameter). The microcirculation in most organsand tissues allows the free passage of these blood cells. Whenmicrothrombii (blood clots) of size greater than 10-15 microns arepresent in circulation, a risk of infarction or blockage of thecapillaries results, leading to ischemia or oxygen deprivation andpossible tissue death. Injection into the circulation of particlesgreater than 10-15 microns in diameter, therefore, must be avoided. Asuspension of particles less than 7-8 microns, is however, relativelysafe and has been used for the delivery of pharmacologically activeagents in the form of liposomes and emulsions, nutritional agents, andcontrast media for imaging applications.

The size of particles and their mode of delivery determines theirbiological behavior. Strand et al. [in Microspheres-BiomedicalApplications, ed. A. Rembaum, pp 193-227, CRC Press (1988)] havedescribed the fate of particles to be dependent on their size. Particlesin the size range of a few nanometers (nm) to 100 nm enter the lymphaticcapillaries following interstitial injection, and phagocytosis may occurwithin the lymph nodes. After intravenous/intraarterial injection,particles less than about 2 microns will be rapidly cleared from theblood stream by the reticuloendothelial system (RES), also known as themononuclear phagocyte system (MPS). Particles larger than about 7microns will, after intravenous injection, be trapped in the lungcapillaries. After intraarterial injection, particles are trapped in thefirst capillary bed reached. Inhaled particles are trapped by thealveolar macrophages.

Pharmaceuticals that are water-insoluble or poorly water-soluble andsensitive to acid environments in the stomach cannot be conventionallyadministered (e.g., by intravenous injection or oral administration).The parenteral administration of such pharmaceuticals has been achievedby emulsification of the oil solubilized drug with an aqueous liquid(such as normal saline) in the presence of surfactants or emulsionstabilizers to produce stable microemulsions. These emulsions may beinjected intravenously, provided the components of the emulsion arepharmacologically inert. U.S. Pat. No. 4,073,943 describes theadministration of water-insoluble pharmacologically active agentsdissolved in oils and emulsified with water in the presence ofsurfactants such as egg phosphatides, pluronics (copolymers ofpolypropylene glycol and polyethylene glycol), polyglycerol oleate, etc.PCT International Publication No. W085/00011 describes pharmaceuticalmicrodroplets of an anaesthetic coated with a phospholipid such asdimyristoyl phosphatidylcholine having suitable dimensions forintradermal or intravenous injection.

Protein microspheres have been reported in the literature as carriers ofpharmacological or diagnostic agents. Microspheres of albumin have beenprepared by either heat denaturation or chemical crosslinking. Heatdenatured microspheres are produced from an emulsified mixture (e.g.,albumin, the agent to be incorporated, and a suitable oil) attemperatures between 100° C. and 150° C. The microspheres are thenwashed with a suitable solvent and stored. Leucuta et al. [InternationalJournal of Pharmaceutics Vol. 41:213-217 (1988)] describe the method ofpreparation of heat denatured microspheres.

The procedure for preparing chemically crosslinked microspheres involvestreating the emulsion with glutaraldehyde to crosslink the protein,followed by washing and storage. Lee et al. [Science Vol. 213:233-235(1981)] and U.S. Pat. No. 4,671,954 teach this method of preparation.

The above techniques for the preparation of protein microspheres ascarriers of pharmacologically active agents, although suitable for thedelivery of water-soluble agents, are incapable of entrappingwater-insoluble ones. This limitation is inherent in the technique ofpreparation which relies on crosslinking or heat denaturation of theprotein component in the aqueous phase of a water-in-oil emulsion. Anyaqueous-soluble agent dissolved in the protein-containing aqueous phasemay be entrapped within the resultant crosslinked or heat-denaturedprotein matrix, but a poorly aqueous-soluble or oil-soluble agent cannotbe incorporated into a protein matrix formed by these techniques.

BRIEF DESCRIPTION OF THE INVENTION

Thus it is an object of this invention to deliver pharmacologicallyactive agents (e.g., taxol, taxane, Taxotere, and the like) inunmodified form in a composition that does not cause allergic reactionsdue to the presence of added emulsifiers and solubilizing agents, as arecurrently employed in drug delivery.

It is a further object of the present invention to deliverpharmacologically active agents in a composition of microparticlessuspended in a suitable biocompatible liquid.

It is yet another object of the invention to deliver pharmacologicallyactive agents enclosed within a polymer shell which is further suspendedin a biocompatible liquid.

These and other objects of the invention will become apparent uponreview of the specification and claims.

In accordance with the present invention, we have discovered thatsubstantially water insoluble pharmacologically active agents can bedelivered in the form of microparticles that are suitable for parenteraladministration in aqueous suspension. This mode of delivery obviates thenecessity for administration of substantially water insolublepharmacologically active agents (e.g., taxol) in an emulsion containing,for example, ethanol and polyethoxylated castor oil, diluted in normalsaline (see, for example, Norton et al., in Abstracts of the 2ndNational Cancer Institute Workshop on Taxol & Taxus, Sep. 23-24, 1992).A disadvantage of such known compositions is their propensity to produceallergic side effects.

The delivery of substantially water insoluble pharmacologically activeagents in the form of a microparticulate suspension allows some degreeof targeting to organs such as the liver, lungs, spleen, lymphaticcirculation, and the like, through the use of particles of varying size,and through administration by different routes. The invention method ofdelivery further allows the administration of substantially waterinsoluble pharmacologically active agents employing a much smallervolume of liquid and requiring greatly reduced administration timerelative to administration volumes and times required by prior artdelivery systems (e.g., intravenous infusion of approximately one to twoliters of fluid over a 24 hour period are required to deliver a typicalhuman dose of 200-400 mg of taxol).

In accordance with another embodiment of the present invention, we havedeveloped compositions useful for in vivo delivery of substantiallywater insoluble pharmacologically active agents. Invention compositionscomprise substantially water insoluble pharmacologically active agents(as a solid or liquid) contained within a polymeric shell. The polymericshell is a biocompatible polymer, crosslinked by the presence ofdisulfide bonds. The polymeric shell, containing substantially waterinsoluble pharmacologically active agents therein, is then suspended ina biocompatible aqueous liquid for administration.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are providedcompositions for in vivo delivery of a substantially water insolublepharmacologically active agent,

wherein said agent is a solid or liquid substantially completelycontained within a polymeric shell,

wherein the largest cross-sectional dimension of said shell is nogreater than about 10 microns,

wherein said polymeric shell comprises a biocompatible polymer which issubstantially crosslinked by way of disulfide bonds, and

wherein said polymeric shell containing pharmacologically active agenttherein is suspended in a biocompatible aqueous liquid.

As used herein, the term "in vivo delivery" refers to delivery of apharmacologically active agent by such routes of administration as oral,intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular,inhalational, topical, transdermal, suppository (rectal), pessary(vaginal), and the like.

As used herein, the term "micron" refers to a unit of measure of oneone-thousandth of a millimeter.

As used herein, the term "biocompatible" describes a substance that doesnot appreciably alter or affect in any adverse way, the biologicalsystem into which it is introduced.

Key differences between the pharmacologically active agents contained ina polymeric shell according to the invention and protein microspheres ofthe prior art are in the nature of formation and the final state of theprotein after formation of the particle, and its ability to carry poorlyaqueous-soluble or substantially aqueous-insoluble agents. In accordancewith the present invention, the polymer (e.g., a protein) is selectivelychemically crosslinked through the formation of disulfide bonds through,for example, the amino acid cysteine that occurs in the naturalstructure of a number of proteins. A sonication process is used todisperse a dispersing agent containing dissolved or suspendedpharmacologically active agent into an aqueous solution of abiocompatible polymer bearing sulfhydryl or disulfide groups (e.g.,albumin) whereby a shell of crosslinked polymer is formed around finedroplets of non-aqueous medium. The sonication process producescavitation in the liquid that causes tremendous local heating andresults in the formation of superoxide ions that crosslink the polymerby oxidizing the sulfhydryl residues (and/or disrupting existingdisulfide bonds) to form new, crosslinking disulfide bonds.

In contrast to the invention process, the prior art method ofglutaraldehyde crosslinking is nonspecific and essentially reactive withany nucleophilic group present in the protein structure (e.g., aminesand hydroxyls). Heat denaturation as taught by the prior artsignificantly and irreversibly alters protein structure. In contrast,disulfide formation contemplated by the present invention does notsubstantially denature the protein. In addition, particles ofsubstantially water insoluble pharmacologically active agents containedwithin a shell differ from crosslinked or heat denatured proteinmicrospheres of the prior art because the polymeric shell produced bythe invention process is relatively thin compared to the diameter of thecoated particle. It has been determined (by transmission electronmicroscopy) that the "shell thickness" of the polymeric coat isapproximately 25 nanometers for a coated particle having a diameter of 1micron (1000 nanometers). In contrast, microspheres of the prior art donot have protein shells, but rather, have protein dispersed throughoutthe volume of the microsphere.

The polymeric shell containing solid or liquid cores ofpharmacologically active agent allows for the delivery of high doses ofthe pharmacologically active agent in relatively small volumes. Thisminimizes patient discomfort at receiving large volumes of fluid andminimizes hospital stay. In addition, the walls of the polymeric shellare generally completely degradable in vivo by proteolytic enzymes(e.g., when the polymer is a protein), resulting in no side effects fromthe delivery system as is the case with current formulations.

According to this embodiment of the present invention, particles ofsubstantially water insoluble pharmacologically active agents arecontained within a shell having a cross-sectional diameter of no greaterthan about 10 microns. A cross-sectional diameter of less than 5 micronsis more preferred, while a cross-sectional diameter of less than 1micron is presently the most preferred for the intravenous route ofadministration.

Substantially water insoluble pharmacologically active agentscontemplated for use in the practice of the present invention includepharmaceutically active agents, diagnostic agents, agents of nutritionalvalue, and the like. Examples of pharmaceutically active agents includetaxol (as used herein, the term "taxol" is intended to include taxolanalogs and prodrugs, taxanes, and other taxol-like drugs, e.g.,Taxotere, and the like), camptothecin and derivatives thereof (whichcompounds have great promise for the treatment of colon cancer),aspirin, ibuprofen, piroxicam, cimetidine, substantially water insolublesteroids (e.g., estrogen, prednisolone, cortisone, hydrocortisone,diflorasone, and the like), drugs such as phenesterine, duanorubicin,doxorubicin, mitotane, visadine, halonitrosoureas, anthrocylines,ellipticine, diazepam, and the like, anaesthetics such asmethoxyfluorane, isofluorane, enfluorane, halothane, benzocaine,dantrolene, barbiturates, and the like. In addition, also contemplatedare substantially water insoluble immunosuppressive agents, such as, forexample, cyclosporines, azathioprine,(17-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone), prednisone, and the like. Apresently preferred pharmaceutically active agent for use in thepractice of the present invention is taxol, which is commerciallyavailable from the manufacturer as needle-like crystals.

Examples of diagnostic agents contemplated for use in the practice ofthe present invention include ultrasound contrast agents, radiocontrastagents (e.g., iodo-octanes, halocarbons, renografin, and the like),magnetic contrast agents (e.g., fluorocarbons, lipid solubleparamagnetic compounds, and the like), as well as other diagnosticagents which cannot readily be delivered without some physical and/orchemical modification to accomodate the substantially water insolublenature thereof.

Examples of agents of nutritional value contemplated for use in thepractice of the present invention include amino acids, sugars, proteins,carbohydrates, fat-soluble vitamins (e.g., vitamins A, D, E, K, and thelike) or fat, or combinations of any two or more thereof.

A number of biocompatible polymers may be employed in the practice ofthe present invention for the formation of the polymeric shell whichsurrounds the substantially water insoluble pharmacologically activeagents. Essentially any polymer, natural or synthetic, bearingsulfhydryl groups or disulfide bonds within its structure may beutilized for the preparation of a disulfide crosslinked shell aboutparticles of substantially water insoluble pharmacologically activeagents. The sulfhydryl groups or disulfide linkages may be preexistingwithin the polymer structure or they may be introduced by a suitablechemical modification. For example, natural polymers such as proteins,oligopeptides, polynucleic acids, polysaccharides (e.g., starch,cellulose, dextrans, alginates, chitosan, pectin, hyaluronic acid, andthe like), and so on, are candidates for such modification.

As examples of suitable biocompatible polymers, naturally occurring orsynthetic proteins may be employed, so long as such proteins havesufficient cysteine residues within their amino acid sequences so thatcrosslinking (through disulfide bond formation, for example, as a resultof oxidation during sonication) can occur. Examples of suitable proteinsinclude albumin (which contains 35 cysteine residues), insulin (whichcontains 6 cysteines), hemoglobin (which contains 6 cysteine residuesper α₂ β₂ unit), lysozyme (which contains 8 cysteine residues),immunoglobulins, α-2-macroglobulin, fibronectin, vitronectin,fibrinogen, and the like.

A presently preferred protein for use in the formation of a polymericshell is albumin. Optionally, proteins such as α-2-macroglobulin, aknown opsonin, could be used to enhance uptake of the shell encasedparticles of substantially water insoluble pharmacologically activeagents by macrophage-like cells, or to enhance the uptake of the shellencased particles into the liver and spleen.

Similarly, synthetic polypeptides containing cysteine residues are alsogood candidates for formation of a shell about the substantially waterinsoluble pharmacologically active agents. In addition, polyvinylalcohol, polyhydroxyethyl methacrylate, polyacrylic acid,polyethyloxazoline, polyacrylamide, polyvinyl pyrrolidinone, and thelike, are good candidates for chemical modification (to introducesulfhydryl and/or disulfide linkages) and shell formation (by causingthe crosslinking thereof).

In the preparation of invention compositions, one can optionally employa dispersing agent to suspend or dissolve the substantially waterinsoluble pharmacologically active agent. Dispersing agents contemplatedfor use in the practice of the present invention include any nonaqueousliquid that is capable of suspending or dissolving the pharmacologicallyactive agent, but does not chemically react with either the polymeremployed to produce the shell, or the pharmacologically active agentitself. Examples include vegetable oils (e.g., soybean oil, coconut oil,olive oil, safflower oil, cotton seed oil, and the like), aliphatic,cycloaliphatic, or aromatic hydrocarbons having 4-30 carbon atoms (e.g.,n-dodecane, n-decane, n-hexane, cyclohexane, toluene, benzene, and thelike), aliphatic or aromatic alcohols having 2-30 carbon atoms (e.g.,octanol, and the like), aliphatic or aromatic esters having 2-30 carbonatoms (e.g., ethyl caprylate (octanoate), and the like), alkyl, aryl, orcyclic ethers having 2-30 carbon atoms (e.g., diethyl ether,tetrahydrofuran, and the like), alkyl or aryl halides having 1-30 carbonatoms (and optionally more than one halogen substituent, e.g., CH₃ Cl,CH₂ Cl₂, CH₂ Cl--CH₂ Cl, and the like), ketones having 3-30 carbon atoms(e.g., acetone, methyl ethyl ketone, and the like), polyalkylene glycols(e.g., polyethylene glycol, and the like), or combinations of any two ormore thereof.

Especially preferred combinations of dispersing agents include volatileliquids such as dichloromethane, ethyl acetate, benzene, and the like(i.e., solvents that have a high degree of solubility for thepharmacologically active agent, and are soluble in the other dispersingagent employed), along with a higher molecular weight (less volatile)dispersing agent. When added to the other dispersing agent, thesevolatile additives help to drive the solubility of the pharmacologicallyactive agent into the dispersing agent. This is desirable since thisstep is usually time consuming. Following dissolution, the volatilecomponent may be removed by evaporation (optionally under vacuum).

Particles of pharmacologically active agent substantially completelycontained within a polymeric shell, prepared as described above, aredelivered as a suspension in a biocompatible aqueous liquid. This liquidmay be selected from water, saline, a solution containing appropriatebuffers, a solution containing nutritional agents such as amino acids,sugars, proteins, carbohydrates, vitamins or fat, and the like.

In accordance with another embodiment of the present invention, there isprovided a method for the preparation of a substantially water insolublepharmacologically active agent for in vivo delivery, said methodcomprising subjecting a mixture comprising:

dispersing agent containing said pharmacologically active agentdispersed therein, and

aqueous medium containing biocompatible polymer capable of beingcrosslinked by disulfide bonds

to sonication conditions for a time sufficient to promote crosslinkingof said biocompatible polymer by disulfide bonds.

A nonobvious feature of the above-described process is in the choice ofdispersing agent, specifically with respect to the polarity of thedispersing agent. The formation of a shell about the particles ofpharmacologically active agent involves unfolding and reorientation ofthe polymer at the interface between the aqueous and non-aqueous phasessuch that the hydrophilic regions within the polymer are exposed to theaqueous phase while the hydrophobic regions within the polymer areoriented towards the non-aqueous phase. In order to effect unfolding ofthe polymer, or change the conformation thereof, energy must be suppliedto the polymer. The interfacial free energy (interfacial tension)between the two liquid phases (i.e., aqueous and non-aqueous)contributes to changes in polymer conformation at that interface.Thermal energy also contributes to the energy pool required forunfolding and/or change of polymer conformation.

Thermal energy input is a function of such variables as the acousticpower employed in the sonication process, the sonication time, thenature of the material being subjected to sonication, the volume of thematerial being subjected to sonication, and the like. The acoustic powerof sonication processes can vary widely, typically falling in the rangeof about 1 up to 1000 watts/cm² ; with an acoustic power in the range ofabout 50 up to 200 watts/cm² being a presently preferred range.Similarly, sonication time can vary widely, typically falling in therange of a few seconds up to about 5 minutes. Preferably, sonicationtime will fall in the range of about 15 up to 60 seconds. Those of skillin the art recognize that the higher the acoustic power applied, theless sonication time is required, and vice versa.

The interfacial free energy is directly proportional to the polaritydifference between the two liquids. Thus at a given operatingtemperature a minimum free energy at the interface between the twoliquids is essential to form the desired polymer shell. Thus, if ahomologous series of dispersing agents is taken with a gradual change inpolarity, e.g., ethyl esters of alkanoic acids, then higher homologuesare increasingly nonpolar, i.e., the interfacial tension between thesedispersing agents and water increases as the number of carbon atoms inthe ester increases. Thus it is found that, although ethyl acetate iswater-immiscible (i.e., an ester of a 2 carbon acid), at roomtemperature (˜20° C.), this dispersing agent alone will not give asignificant yield of polymer shell-coated particles. In contrast, ahigher ester such as ethyl octanoate (ester of an 8 carbon acid) givespolymer shell-coated particles in high yield. In fact, ethyl heptanoate(ester of a 7 carbon acid) gives a moderate yield while the lower esters(esters of 3, 4, 5, or 6 carbon acids) give poor yield. Thus, at a giventemperature, one could set a condition of minimum aqueous-dispersingagent interfacial tension required for formation of high yields ofpolymer shell-coated particles.

Temperature is another variable that may be manipulated to affect theyield of polymer shell-coated particles. In general the surface tensionof a liquid decreases with increasing temperature. The rate of change ofsurface tension with temperature is often different for differentliquids. Thus, for example, the interfacial tension (Δγ) between twoliquids may be Δγ₁ at temperature T₁ and Δγ₂ at temperature T₂. If Δγ₁at T₁ is close to the minimum required to form polymeric shells of thepresent invention, and if Δγ₂ (at temp. T₂) is greater than Δγ₁, then achange of temperature from T₁ to T₂ will increase the yield of polymericshells. This, in fact, is observed in the case of ethyl heptanoate,which gives a moderate yield at 20° C. but gives a high yield at 10° C.

Temperature also affects the vapor pressure of the liquids employed. Thelower the temperature, the lower the total vapor pressure. The lower thetotal vapor pressure, the more efficient is the collapse of thecavitation bubble. A more efficient collapse of the sonication bubblecorrelates with an increased rate of superoxide (HO₂ ⁻) formation.Increased rate of superoxide, formation leads to increased yields ofpolymeric shells at lower temperatures. As a countervailingconsideration, however, the reaction rate for oxidation of sulfhydrylgroups (i.e., to form disulfide linkages) by superoxide ions increaseswith increasing temperature. Thus for a given liquid subjected tosonication conditions, there exists a fairly narrow range of optimumoperating temperatures within which a high yield of polymeric shells isobtained.

Thus a combination of two effects, i.e., the change in surface tensionwith temperature (which directly affects unfolding and/or conformationalchanges of the polymer) and the change in reaction yield (the reactionbeing crosslinking of the polymer via formation of disulfide linkages)with temperature dictate the overall conversion or yield of polymershell-coated particles.

The sonication process described above may be manipulated to producepolymer shell-coated particles containing pharmacologically active agenthaving a range of sizes. Presently preferred particle radii fall in therange of about 0.1 up to about 5 micron. A narrow size distribution inthis range is very suitable for intravenous drug delivery. The polymershell-coated particles are then suspended in an aqueous biocompatibleliquid (as described above) prior to administration by suitable means.

Variations on the general theme of dissolved pharmacologically activeagent enclosed within a polymeric shell are possible. A suspension offine particles of pharmacologically active agent in a biocompatibledispersing agent could be used (in place of a biocompatible dispersingagent containing dissolved pharmacologically active agent) to produce apolymeric shell containing dispersing agent-suspended pharmacologicallyactive agent particles. In other words, the polymeric shell couldcontain a saturated solution of pharmacologically active agent indispersing agent. Another variation is a polymeric shell containing asolid core of pharmacologically active agent produced by initiallydissolving the pharmacologically active agent in a volatile organicsolvent (e.g. benzene), forming the polymeric shell and evaporating thevolatile solvent under vacuum, e.g., in a rotary evaporator, orfreeze-drying the entire suspension. This results in a structure havinga solid core of pharmacologically active agent surrounded by a polymercoat. This latter method is particularly advantageous for deliveringhigh doses of pharmacologically active agent in a relatively smallvolume. In some cases, the polymer forming the shell about the corecould itself be a therapeutic or diagnostic agent, e.g., in the case ofinsulin, which may be delivered as part of a polymeric shell formed inthe sonication process described above.

Variations in the polymeric shell are also possible. For example, asmall amount of PEG containing sulfhydryl groups could be included withthe polymer. Upon sonication, the PEG is crosslinked into the polymerand forms a component of the polymeric shell. PEG is known for itsnonadhesive character and has been attached to proteins and enzymes toincrease their circulation time in vivo [Abuchowski et al., J. Biol.Chem. Vol. 252:3578 (1977)]. It has also been attached to phospholipidsforming the lipidic bilayer in liposomes to reduce their uptake andprolong lifetimes in vivo [Klibanov et al., FEBS Letters Vol. 268:235(1990)]. Thus the incorporation of PEG into the walls of crosslinkedprotein shells alters their blood circulation time. This property can beexploited to maintain higher blood levels of the pharmacologicallyactive agent and prolonged pharmacologically active agent release times.

One skilled in the art will recognize that several variations arepossible within the scope and spirit of this invention. The dispersingagent within the polymeric shell may be varied, a large variety ofpharmacologically active agents may be utilized, and a wide range ofproteins as well as other natural and synthetic polymers may be used inthe formation of the walls of the polymeric shell. Applications are alsofairly wide ranging. Other than biomedical applications such as thedelivery of drugs, diagnostic agents (in imaging applications),artificial blood (sonochemically crosslinked hemoglobin) and parenteralnutritional agents, the polymeric shell structures of the invention maybe incorporated into cosmetic applications such as skin creams or haircare products, in perfumery applications, in pressure sensitive inks,and the like.

An approach to the problem of taxol administration that has not beendescribed in the literature is its delivery as an aqueous suspension ofmicron size particles, or an aqueous suspension containing eitherparticles of taxol or taxol dissolved in a biocompatible non-aqueousliquid. This approach would facilitate the delivery of taxol atrelatively high concentrations and obviate the use of emulsifiers andtheir associated toxic side effects.

In accordance with yet another embodiment of the present invention, theabove-described mode of administration is facilitated by noveltaxol-containing compositions wherein taxol is suspended in abiocompatible liquid, and wherein the resulting suspension containsparticles of taxol having a cross-sectional dimension no greater thanabout 10 microns. The desired particle size of less than about 10microns can be achieved in a variety of ways, e.g., by grinding, spraydrying, precipitation, sonication, and the like.

Due to the crystal size of conventionally obtained taxol which isgreater than 20 microns, solid particles of taxol have not beendelivered in the form of a suspension in a vehicle such as normalsaline. However, the present invention discloses the delivery of aparticulate suspension of taxol ground to a size less than 10 microns,preferably less than 5 microns and most preferably less than 1 micron,which allows intravenous delivery in the form of a suspension withoutthe risk of blockage in the microcirculation of organs and tissues.

Due to the microparticular nature of the delivered drug, most of it iscleared from the circulation by organs having reticuloendothelialsystems such as the spleen, liver, and lungs. This allowspharmacologically active agents in particulate form to be targeted tosuch sites within the body.

Biocompatible liquids contemplated for use in this embodiment are thesame as those described above. In addition, parenteral nutritionalagents such as Intralipid (trade name for a commercially available fatemulsion used as a parenteral nutrition agent; available from KabiVitrum, Inc., Clayton, N.C.), Nutralipid (trade name for a commerciallyavailable fat emulsion used as a parenteral nutrition agent; availablefrom McGaw, Irvine, Calif.), Liposyn III (trade name for a commerciallyavailable fat emulsion used as a parenteral nutrition agent (containing20% soybean oil, 1.2% egg phosphatides, and 2.5% glycerin); availablefrom Abbott Laboratories, North Chicago, Ill.), and the like may be usedas the carrier of the drug particles. Alternatively, if thebiocompatible liquid contains a drug-solubilizing material such assoybean oil (e.g., as in the case of Intralipid), the drug may bepartially or completely solubilized within the carrier liquid, aidingits delivery. An example of such a case is the delivery of taxol inIntralipid as the carrier. Presently preferred biocompatible liquids foruse in this embodiment are parenteral nutrition agents, such as thosedescribed above.

In accordance with still another embodiment of the present invention,there is provided a composition for the in vivo delivery of taxolwherein taxol is dissolved in a parenteral nutrition agent.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Preparation of Taxol Particles

Crystals of taxol (Sigma Chemical) were ground in a ball mill untilparticles of solid taxol were obtained having a size less than 10microns. Size of particles were determined by suspending the particlesin isotonic saline and counting with the aid of a particle counter(Elzone, Particle Data). Grinding was continued until 100% of theparticles had a size less than 5 microns. The preferred particle sizefor intravenous delivery is less than 5 microns and most preferably lessthan 1 micron.

Alternatively, particles of taxol were obtained by sonicating asuspension of taxol in water until all particles were below 10 microns.

Taxol particles less than 10 microns can also be obtained byprecipitating taxol from a solution of taxol in ethanol by adding wateruntil a cloudy suspension is obtained. Optionally, the solution of taxolcan be sonicated during the water addition, until a cloudy suspension isobtained. The resulting suspension is then filtered and dried to obtainpure taxol particles in the desired size range.

Fine particles of taxol were prepared by spray drying a solution oftaxol in a volatile organic such as ethanol. The solution was passedthrough an ultrasonic nozzle that formed droplets of ethanol containingtaxol. As the ethanol evaporated in the spray drier, fine particles oftaxol were obtained. Particle size can be varied by changing theconcentration of taxol in ethanol, adjusting the flow rate of liquidthrough the nozzle and power of sonication.

EXAMPLE 2 Preparation of Protein Shell Containing Oil

Three ml of a USP (United States Pharmacopeia) 5% human serum albuminsolution (Alpha Therapeutic Corporation) were taken in a cylindricalvessel that could be attached to a sonicating probe (Heat Systems, ModelXL2020). The albumin solution was overlayered with 6.5 ml of USP gradesoybean oil (soya oil). The tip of the sonicator probe was brought tothe interface between the two solutions and the assembly was maintainedin a cooling bath at 20° C. The system was allowed to equilibriate andthe sonicator turned on for 30 seconds. Vigorous mixing occurred and awhite milky suspension was obtained. The suspension was diluted 1:5 withnormal saline. A particle counter (Particle Data Systems, Elzone, Model280 PC) was utilized to determine size distribution and concentration ofoil-containing protein shells. The resulting protein shells weredetermined to have a maximum cross-sectional dimension of about1.35±0.73 microns, and the total concentration determined to be ˜10⁹shells/ml in the original suspension.

EXAMPLE 3 Parameters Affecting Polymeric Shell Formation

Several variables such as protein concentration, temperature, sonicationtime, concentration of pharmacologically active agent, and acousticintensity were tested to optimize formation of polymeric shell. Theseparameters were determined for crosslinked bovine serum albumin shellscontaining toluene.

Polymeric shells made from solutions having protein concentrations of1%, 2.5%, 5% and 10% were counted with the particle counter to determinea change in the size and number of polymeric shells produced. The sizeof the polymeric shells was found not to vary with proteinconcentration, but the number of polymeric shells per ml of "milkysuspension" formed increased with the increase in concentration of theprotein up to 5%. No significant change in the number of polymericshells was found to occur above that concentration.

Initial vessel temperatures were found to be important for optimalpreparation of polymeric shells. Typically, initial vessel temperatureswere maintained between 0° C. and 45° C. The aqueous-oil interfacialtension of the oils used for formation of the polymeric shell was animportant parameter, which also varied as a function of temperature. Theconcentration of pharmacologically active agent was found not tosignificantly effect the yield of protein shells. It is relativelyunimportant if the pharmacologically active agent is incorporated in thedissolved state, or suspended in the dispersing medium.

Sonication time was an important factor determining the number ofpolymeric shells produced per ml. It was found that a sonication timegreater than three minutes produced a decrease in the overall count ofpolymeric shells, indicating possible destruction of polymeric shellsdue to excessive sonication. Sonication times less than three minuteswere found to produce adequate numbers of polymeric shells.

According to the nomograph provided by the manufacturer of thesonicator, the acoustic power rating of the sonicator employed herein isapproximately 150 watts/cm². Three power settings in order of increasingpower were used, and it was found that the maximum number of polymericshells were produced at the highest power setting.

EXAMPLE 4 Preparation of Polymeric Shells Containing Dissolved Taxol

Taxol was dissolved in USP grade soybean oil at a concentration of 2mg/ml. 3 ml of a USP 5% human serum albumin solution was taken in acylindrical vessel that could be attached to a sonicating probe. Thealbumin solution was overlayered with 6.5 ml of soybean oil/taxolsolution. The tip of the sonicator probe was brought to the interfacebetween the two solutions and the assembly was maintained in equilibriumand the sonicator turned on for 30 seconds. Vigorous mixing occurred anda stable white milky suspension was obtained which containedprotein-walled polymeric shells enclosing the oil/taxol solution.

In order to obtain a higher loading of drug into the crosslinked proteinshell, a mutual solvent for the oil and the drug (in which the drug hasa considerably higher solubility) can be mixed with the oil. Providedthis solvent is relatively non-toxic (e.g., ethyl acetate), it may beinjected along with the original carrier. In other cases, it may beremoved by evaporation of the liquid under vacuum following preparationof the polymeric shells.

EXAMPLE 5 Stability of Polymeric Shells

Suspensions of polymeric shells at a known concentration were analyzedfor stability at three different temperatures (i.e., 4° C., 25° C., and38° C.) Stability was measured by the change in particle counts overtime. Crosslinked protein (albumin) shells containing soybean oil (SBO)were prepared as described above (see Example 2), diluted in saline to afinal oil concentration of 20% and stored at the above temperatures.Particle counts (Elzone) obtained for each of the samples as a functionof time are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Protein Shells (#/ml · 10.sup.10)                                    in saline                                                                     Day     4° C. 25° C.                                                                          38° C.                                   ______________________________________                                        0       7.9          8.9      8.1                                             1       7.4          6.9      6.8                                             7       7.3          8.3      5.0                                             9       7.8          8.1      5.8                                             17      7.8          8.3      6.1                                             23      6.9          7.8      7.4                                             27      7.2          8.8      7.1                                             ______________________________________                                    

As demonstrated by the above data, the concentration of countedparticles (i.e., polymeric shells) remains fairly constant over theduration of the experiment. The range is fairly constant and remainsbetween about 7-9.10¹⁰ /ml, indicating good polymeric shell stabilityunder a variety of temperature conditions over almost four weeks.

EXAMPLE 6 In Vivo Biodistribution-Crosslinked Protein Shells Containinga Fluorophore

To determine the fate of crosslinked albumin shells followingintravenous injection, a fluorescent dye (rubrene, obtained fromAldrich) was dissolved in toluene, and crosslinked albumin shellscontaining toluene/rubrene were prepared as described above bysonication. The resulting milky suspension was diluted five times innormal saline. Two ml of the diluted suspension was then injected intothe tail vein of a rat over 10 minutes. One animal was sacrificed anhour after injection and another 24 hours after injection.

Frozen lung, liver, kidney, spleen, and bone marrow sections wereexamined under fluorescence for the presence of polymeric shellscontaining fluorescent dye. At one hour, most of the polymeric shellswere intact and found in the lungs and liver as brightly fluorescingparticles of about 1 micron diameter. At 24 hours, polymeric shells werefound in the liver, lungs, spleen, and bone marrow. A general stainingof the tissue was also observed, indicating that the polymeric shellshad been digested, and the dye liberated from within. This result wasconsistent with expectations and demonstrates the potential use ofinvention compositions for delayed or controlled release of entrappedpharmaceutical agent such as taxol.

EXAMPLE 7 Toxicity of Polymeric Shells Containing Soybean Oil (SBO)

Polymeric shells containing soybean oil were prepared as described inExample 2. The resulting suspension was diluted in normal saline toproduce two different solutions, one containing 20% SBO and the othercontaining 30% SBO.

Intralipid, a commercially available TPN agent, contains 20% SBO. TheLD₅₀ for Intralipid in mice is 120 ml/kg, or about 4 ml for a 30 gmouse, when injected at 1 cc/min.

Two groups of mice (three mice in each group; each mouse weighing about30 g) were treated with invention composition containing SBO as follows.Each mouse was injected with 4 ml of the prepared suspension ofSBO-containing polymeric shells. Each member of one group received thesuspension containing 20% SBO, while each member of the other groupreceive the suspension containing 30% SBO.

All three mice in the group receiving the suspension containing 20% SBOsurvived such treatment, and showed no gross toxicity in any tissues ororgans when observed one week after SBO treatment. Only one of the threemice in the group receiving suspension containing 30% SBO died afterinjection. These results clearly demonstrate that oil contained withinpolymeric shells according to the present invention is not toxic at itsLD₅₀ dose, as compared to a commercially available SBO formulation(Intralipid). This effect can be attributed to the slow release (i.e.,controlled rate of becoming bioavailable) of the oil from within thepolymeric shell. Such slow release prevents the attainment of a lethaldose of oil, in contrast to the high oil dosages attained withcommercially available emulsions.

EXAMPLE 8 In vivo Bioavailability of Soybean Oil Released from PolymericShells

A test was performed to determine the slow or sustained release ofpolymeric shell-enclosed material following the injection of asuspension of polymeric shells into the blood stream of rats.Crosslinked protein (albumin) walled polymeric shells containing soybeanoil (SBO) were prepared by sonication as described above. The resultingsuspension of oil-containing polymeric shells was diluted in saline to afinal suspension containing 20% oil. Five ml of this suspension wasinjected into the cannulated external jugular vein of rats over a 10minute period. Blood was collected from these rats at several timepoints following the injection and the level of triglycerides (soybeanoil is predominantly triglyceride) in the blood determined by routineanalysis.

Five ml of a commercially available fat emulsion (Intralipid, an aqueousparenteral nutrition agent--containing 20% soybean oil, 1.2% egg yolkphospholipids, and 2.25% glycerin) was used as a control. The controlutilizes egg phosphatide as an emulsifier to stabilize the emulsion. Acomparison of serum levels of the triglycerides in the two cases wouldgive a direct comparison of the bioavailability of the oil as a functionof time. In addition to the suspension of polymeric shells containing20% oil, five ml of a sample of oil-containing polymeric shells insaline at a final concentration of 30% oil was also injected. Two ratswere used in each of the three groups. The blood levels of triglyceridesin each case are tabulated in Table 2, given in units of mg/dl.

                  TABLE 2                                                         ______________________________________                                                 SERUM TRIGLYCERIDES (mg/dl)                                          GROUP      Pre     1 hr    4 hr 24 hr 48 hr 72 hr                             ______________________________________                                        Intralipid Control                                                                       11.4    941.9   382.9                                                                              15.0   8.8  23.8                              (20% SBO)                                                                     Polymeric Shells                                                                         24.8     46.7    43.8                                                                              29.3  24.2  43.4                              (20% SBO)                                                                     Polymeric Shells                                                                         33.4     56.1   134.5                                                                              83.2  34.3  33.9                              (30% SBO)                                                                     ______________________________________                                    

Blood levels before injection are shown in the column marked `Pre`.Clearly, for the Intralipid control, very high triglyceride levels areseen following injection. Triglyceride levels are then seen to takeabout 24 hours to come down to preinjection levels. Thus the oil is seento be immediately available for metabolism following injection.

The suspension of oil-containing polymeric shells containing the sameamount of total oil as Intralipid (20%) show a dramatically differentavailability of detectible triglyceride in the serum. The level rises toabout twice its normal value and is maintained at this level for manyhours, indicating a slow or sustained release of triglyceride into theblood at levels fairly close to normal. The group receivingoil-containing polymeric shells having 30% oil shows a higher level oftriglycerides (concomitant with the higher administered dose) that fallsto normal within 48 hours. Once again, the blood levels of triglyceridedo not rise astronomically in this group, compared to the control groupreceiving Intralipid. This again, indicates the slow and sustainedavailability of the oil from invention composition, which has theadvantages of avoiding dangerously high blood levels of materialcontained within the polymeric shells and availability over an extendedperiod at acceptable levels. Clearly, drugs delivered within polymericshells of the present invention would achieve these same advantages.

Such a system of soybean oil-containing polymeric shells could besuspended in an aqueous solution of amino acids, essential electrolytes,vitamins, and sugars to form a total parenteral nutrition (TPN) agent.Such a TPN cannot be formulated from currently available fat emulsions(e.g., Intralipid) due to the instability of the emulsion in thepresence of electrolytes.

EXAMPLE 9 Preparation of Crosslinked Protein-walled Polymeric ShellsContaining a Solid Core of Pharmaceutically Active Agent

Another method of delivering a poorly water-soluble drug such as taxolwithin a polymeric shell is to prepare a shell of polymeric materialaround a solid drug core. Such a `protein coated` drug particle may beobtained as follows. The procedure described in Example 4 is repeatedusing an organic solvent to dissolve taxol at a relatively highconcentration. Solvents generally used are organics such as benzene,toluene, hexane, ethyl ether, and the like. Polymeric shells areproduced as described in Example 4. Five ml of the milky suspension ofpolymeric shells containing dissolved taxol are diluted to 10 ml innormal saline. This suspension is placed in a rotary evaporator at roomtemperature and the volatile organic removed by vacuum. After about 2hours in the rotary evaporator, these polymeric shells are examinedunder a microscope to reveal opaque cores, indicating removal ofsubstantially all organic solvent, and the presence of solid taxolwithin a shell of protein.

Alternatively, the polymeric shells with cores of organicsolvent-containing dissolved drug are freeze-dried to obtain a drycrumbly powder that can be resuspended in saline (or other suitableliquid) at the time of use. In case of other drugs that may not be inthe solid phase at room temperature, a liquid core polymeric shell isobtained. This method allows for the preparation of a crosslinkedprotein-walled shell containing undiluted drug within it. Particle sizeanalysis shows these polymeric shells to be smaller than thosecontaining oil. Although the presently preferred protein for use in theformation of the polymeric shell is albumin, other proteins such asα-2-macroglobulin, a known opsonin, could be used to enhance uptake ofthe polymeric shells by macrophage-like cells. Alternatively, aPEG-sulfhydryl (described below) could be added during formation of thepolymeric shell to produce a polymeric shell with increased circulationtime in vivo.

EXAMPLE 10 In vivo Circulation and Release Kinetics of Polymeric Shells

Solid core polymeric shells containing taxol were prepared as describedabove (see, for example, Example 4) and suspended in normal saline. Theconcentration of taxol in the suspension was measured by HPLC asfollows. First, the taxol within the polymeric shell was liberated bythe addition of 0.1M mercaptoethanol (resulting in exchange of proteindisulfide crosslinkages, and breakdown of the crosslinking of thepolymeric shell), then the liberated taxol was extracted from thesuspension with acetonitrile. The resulting mixture was centrifuged andthe supernatant freeze-dried. The lyophilate was dissolved in methanoland injected onto an HPLC to determine the concentration of taxol in thesuspension. The taxol concentration was found to be about 1.6 mg/ml.

Rats were injected with 2 ml of this suspension through a jugularcatheter. The animal was sacrificed at two hours, and the amount oftaxol present in the liver determined by HPLC. This requiredhomogenization of the liver, followed by extraction with acetonitrileand lyophilization of the supernatant following centrifugation. Thelyophilate was dissolved in methanol and injected onto an HPLC.Approximately 15% of the administered dose of taxol was recovered fromthe liver at two hours, indicating a significant dosage to the liver.This result is consistent with the known function of thereticuloendothelial system of the liver in clearing small particles fromthe blood.

EXAMPLE 11 Preparation of Crosslinked PEG-walled Polymeric Shells

As an alternative to the use of thiol (sulfhydryl) containing proteinsin the formation of, or as an additive to polymeric shells of theinvention, a thiol-containing PEG was prepared. PEG is known to benontoxic, noninflammatory, nonadhesive to cells, and in generalbiologically inert. It has been bound to proteins to reduce theirantigenicity and to liposome forming lipids to increase theircirculation time in vivo. Thus incorporation of PEG into an essentiallyprotein shell would be expected to increase circulation time as well asstability of the polymeric shell. By varying the concentration ofPEG-thiol added to the 5% albumin solution, it was possible to obtainpolymeric shells with varying stabilities in vivo. PEG-thiol wasprepared by techniques available in the literature (such as thetechnique of Harris and Herati, as described in Polymer Preprints Vol.32:154-155 (1991)).

PEG-thiol of molecular weight 2000 g/mol was dissolved at aconcentration of 1% (0.1 g added to 10 ml) in a 5% albumin solution.This protein/PEG solution was overlayered with oil as described inExample 2 and sonicated to produce oil-containing polymeric shells withwalls comprising crosslinked protein and PEG. These Polymeric shellswere tested for stability as described in Example 5.

Other synthetic water-soluble polymers that may be modified with thiolgroups and utilized in lieu of PEG include, for example, polyvinylalcohol, polyhydroxyethyl methacrylate, polyacrylic acid,polyethyloxazoline, polyacrylamide, polyvinyl pyrrolidinone,polysaccharides (such as chitosan, alginates, hyaluronic acid, dextrans,starch, pectin, etc), and the like.

EXAMPLE 12 Targeting of Immunosuppressive Agent to Transplanted Organsusing Intravenous Delivery of Polymeric Shells Containing Such Agents

Immunosuppressive agents are extensively used following organtransplantation for the prevention of rejection episodes. In particular,cyclosporine, a potent immunosuppressive agent, prolongs the survival ofallogeneic transplants involving skin, heart, kidney, pancreas, bonemarrow, small intestine, and lung in animals. Cyclosporine has beendemonstrated to suppress some humoral immunity and to a greater extent,cell mediated reactions such as allograft rejection, delayedhypersensitivity, experimental allergic encephalomyelitis, Freund'sadjuvant arthritis, and graft versus host disease in many animal speciesfor a variety of organs. Successful kidney, liver and heart allogeneictransplants have been performed in humans using cyclosporine.

Cyclosporine is currently delivered in oral form either as capsulescontaining a solution of cyclosporine in alcohol, and oils such as cornoil, polyoxyethylated glycerides and the like, or as a solution in oliveoil, polyoxyethylated glycerides, etc. It is also administered byintravenous injection, in which case it is dissolved in a solution ofethanol (approximately 30%) and Cremaphor (polyoxyethylated castor oil)which must be diluted 1:20 to 1:100 in normal saline or 5% dextroseprior to injection. Compared to an intravenous (i.v.) infusion, theabsolute bioavailibility of the oral solution is approximately 30%(Sandoz Pharmaceutical Corporation, Publication SDI-Z10 (A4), 1990). Ingeneral, the i.v. delivery of cyclosporine suffers from similar problemsas the currently practiced i.v. delivery of taxol, i.e., anaphylacticand allergic reactions believed to be due to the Cremaphor, the deliveryvehicle employed for the i.v. formulation.

In order to avoid problems associated with the Cremaphor, cyclosporinecontained within polymeric shells as described above may be delivered byi.v. injection. It may be dissolved in a biocompatible oil or a numberof other solvents following which it may be dispersed into polymericshells by sonication as described above. In addition, an importantadvantage to delivering cyclosporine (or other immunosuppressive agent)in polymeric shells has the advantage of local targeting due to uptakeof the injected material by the RES system in the liver. This may, tosome extent, avoid systemic toxicity and reduce effective dosages due tolocal targeting. The effectiveness of delivery and targeting to theliver of taxol contained within polymeric shells following intravenousinjection is demonstrated in Example 9. A similar result would beexpected for the delivery of cyclosporine (or other putativeimmunosuppressive agent) in accordance with the present invention.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

That which is claimed is:
 1. A method for the preparation of asubstantially water insoluble pharmacologically active agent for in vivodelivery, said method comprising subjecting a mixture comprising:adispersing agent containing said pharmacologically active agentdispersed therein, and aqueous medium containing a biocompatible polymercapable of being crosslinked by disulfide bonds to sonication conditionsfor a time sufficient to promote crosslinking of said biocompatiblepolymer by disulfide bonds to produce a polymeric shell containing thepharmacologically active agent therein.
 2. A method for the preparationof substantially water insoluble pharmaceutical agents for in vivodelivery, said method comprising subjecting taxol and suitable medium tosonication conditions for a time sufficient to produce particles havinga maximum cross-sectional dimension of no greater than about 10 microns.3. The method according to claim 1 wherein said dispersing agent isselected from soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, aliphatic, cycloaliphatic or aromatic hydrocarbons having 4-30carbon atoms, aliphatic or aromatic alcohols having 2-30 carbon atoms,aliphatic or aromatic esters having 2-30 carbon atoms, alkyl, aryl, orcyclic ethers having 2-30 carbon atoms, alkyl or aryl halides having1-30 carbon atoms, optionally having more than one halogen substituent,ketones having 3-30 carbon atoms, polyalkylene glycol, or combinationsof any two or more thereof.
 4. The method according to claim 3 whereinsaid dispersing agent comprises a volatile dispersing agent.
 5. Themethod according to claim 4 wherein the volatile dispersing agent isselected from benzene, toluene, hexane, ethyl ether, dichloromethane, orethyl acetate.
 6. The method according to claim 1 wherein saidsubstantially water insoluble pharmacologically active agent is selectedfrom a pharmaceutically active agent, a diagnostic agent, or an agent ofnutritional value.
 7. The method according to claim 6 wherein saidpharmaceutically active agent is selected from taxol, taxotere,campothecin, aspirin, ibuprofen, piroxicam, cimetidine, substantiallywater insoluble steroids, phenesterine, duanorubicin, doxorubicin,mitotane, visadine, halonitrosoureas, anthrocylines, ellipticine,diazepam, methoxyfluorane, isofluorane, enfluorane, halothane,benzocaine, dantrolene, or barbiturates.
 8. The method according toclaim 6 where said pharmaceutically active agent is a substantiallywater insoluble immunosuppressive agent selected from cyclosporines,azathioprine,17-ally-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9]octacos-18-ene-2,3,10,16-tetraone, or prednisone.
 9. The methodaccording to claim 6 wherein said diagnostic agent is selected fromultrasound contrast agents radiocontrast agents, or magnetic contrastagents.
 10. The method according to claim 9 wherein said radiocontrastagent is selected from iodo-octanes, halocarbons, or renografin.
 11. Themethod according to claim 9 wherein said magnetic contrast agent is alipid soluble paramagnetic compound.
 12. The method according to claim 6wherein said agent of nutritional value is selected from amino acids,sugars, proteins, carbohydrates, fat-soluble vitamins, or fat, orcombinations of any two or more thereof.
 13. The method according toclaim 12 wherein said pharmacologically active agent within said shellis dissolved in a biocompatible dispersing agent.
 14. The methodaccording to claim 1 wherein said pharmacologically active agent withinsaid shell is suspended in a biocompatible dispersing agent.
 15. Themethod according to claim 1 further comprising suspending the polymericshells in a biocompatible aqueous liquid.
 16. The method according toclaim 15 wherein said biocompatible aqueous liquid is selected fromwater, saline, a solution containing appropriate buffers, or a solutioncontaining nutritional agents.
 17. The method according to claim 1wherein said biocompatible polymer is a naturally occurring polymer, asynthetic polymer, or a combination thereof,wherein said polymer, priorto crosslinking, has covalently attached thereto sulfhydryl groups ordisulfide linkages.
 18. The method according to claim 17 wherein saidnaturally occurring polymers are selected from proteins, lipids,polynucleic acids or polysaccharides.
 19. The method according to claim18 wherein said protein is selected from albumin, insulin, hemoglobin,lysozyme, immunoglobulins, alpha-2-macroglobulin, fibronectin,vitronectin, or fibrinogen.
 20. The method according to claim 19 whereinsaid protein is albumin.
 21. The method according to claim 18 whereinsaid polysaccharide is selected from starch, cellulose, dextrans,alginates, chitosan, pectin, or hyaluronic acid.
 22. The methodaccording to claim 17 wherein said synthetic polymers are selected fromsynthetic polyamino acids containing cysteine residues and/or disulfidegroups, polyvinyl alcohol modified to contain free sulfhydryl groupsand/or disulfide groups, polyhydroxyethyl methacrylate modified tocontain free sulfhydryl groups and/or disulfide groups, polyacrylic acidmodified to contain free sulfhydryl groups and/or disulfide groups,polyethyloxazoline modified to contain free sulfhydryl groups and/ordisulfide groups, polyacrylamide modified to contain free sulfhydrylgroups and/or disulfide groups, polyvinyl pyrrolidone modified tocontain free sulfhydryl groups and/or disulfide groups, polyalkyleneglycols modified to contain free sulfhydryl groups and/or disulfidegroups, as well as mixtures of any two or more thereof.
 23. The methodaccording to claim 1 wherein the mixture is subjected to sonicationconditions comprising acoustic power in the range of 1 up to 1000watts/cm².
 24. The method according to claim 1 wherein the mixture issubjected to sonication conditions comprising acoustic power in therange of 50 up to 500 watts/cm².
 25. The method according to claim 1wherein the mixture is subjected to sonication for less than 5 minutes.26. The method according to claim 1 wherein the mixture is subjected tosonication for a time ranging from 15 to 60 seconds.
 27. The methodaccording to claim 1 further comprising removing the dispersing agentfrom the mixture.
 28. The method according to claim 1 wherein thelargest cross-sectional dimension of said shell is no greater than about10 microns.